Antibodies that bind LDCAM

ABSTRACT

The invention is directed to LDCAM as a purified and isolated protein, the DNA encoding the LDCAM, host cells transfected with cDNAs encoding LDCAM, processes for preparing LDCAM polypeptides and compositions and methods for treating utilizing LDCAM polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation is a continuation of U.S. patentapplication Ser. No. 10/622,237, filed Jul. 17, 2003, which is acontinuation of U.S. patent application Ser. No. 09/778,187, filed Feb.6, 2001, which is a continuation-in-part of International ApplicationNo. PCT/US99/17905, filed 05 Aug. 1999, which was published under PCTArticle 21(2) on 17 Feb. 2000, in English, as WO 00/08158, and whichclaims the benefit of U.S. Provisional Application Ser. No. 60/095,672,filed 07 Aug. 1998, all of which are incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates to new molecules, designated LDCAM,capable of modulating or altering T cell function. More particularly,the present invention involves novel polypeptides that interact with Tcell surface molecules to alter signaling, bind to themselves and bindto another novel polypeptide, designated B7L-1, and generate increasesin natural killer cell populations. The invention includes LDCAMmolecules, DNA encoding LDCAM molecules, processes for production ofrecombinant LDCAM polypeptides, and pharmaceutical compositionscontaining such LDCAM polypeptides.

2. Description of Related Art

Adhesion molecules play important roles in cell signaling within theimmune system and other cellular systems. In addition to the antigenspecific signals delivered by the T cell receptor complex, the shape andtype of immune response by T cells depend upon costimulatory signalsmediated by adhesion molecules on antigen presenting cells (APC). Onesuch costimulatory signaling involves the adhesion molecules B7-1 (CD80)and B7-2 (CD86) which send important signals through their T cellsurface receptors, CD28 and CTLA4 (CD152). B7-1 interacts with CD28 tosignal cytokine production, cell proliferation, and the generation ofeffector and memory T cells. If the signal through CD28 is blocked, Tcell anergy or immune deviation can occur, resulting in severelydepressed or altered immune responses.

B7-1 also interacts with the T cell CTLA4 receptor. Its signaling iscomplex, but one component provides a negative feedback signal, causingthe T cell to attenuate the CD28 signal. In the absence of this signal,rampant T cell proliferation and effector cell activation continues.When this feedback regulation malfunctions, autoimmune diseases andlymphoproliferation can result. For example, when the CD28 and B7-1 (andB7-2) interaction is blocked with an anti-CTLA4 antibody, increasedtumor immunity and lymphoproliferation have been observed.

B7-2, which is expressed on different cells and at different stages ofAPC activation from that of B7-1, also delivers its costimulatory signalto T cells through CD28 and CTLA4. The B7-2 signal can lead to immuneresponses that are identical to, or different from the immune responsesresulting from B7-1 signaling. The nature of the B7-2 signaling dependsupon the cellular context and the timing of the costimulation.

Even though they bind to the same cellular receptors, B7-1 and B7-2 areonly weakly related at the amino acid level. Both, however, are membersof the extended immunoglobulin domain containing superfamily and much oftheir shared sequence homology is due to the particular residues sharedby their common Ig domains, which are characteristic of the Ig-domainsubfamily.

There is evidence to suggest that other adhesion molecules are importantin T cell response to antigens. For example, T cell proliferation andcytokine production that occurs in response to engagement of a T cellreceptor by an antigen can occur in the absence of CD28 in certaindiseases. Proliferation and cytokine production also occurs in theabsence of CD28 in memory responses, and in systems in which CD28 hasbeen genetically removed. In some cases, T cell proliferation dependsupon an interaction within the CD48 or the ICAM/LFA systems.Furthermore, the adhesion molecule known as ALCAM interacts with its Tcell ligand CD6 to modulate the CD3 signal.

Clearly, signaling through T cell surface receptors plays an importantrole in maintaining balance in the immune system. Systems with apredominance of activatory signals, such as the costimulatory signalingbetween CD28 and B7-1, can lead to autoimmunity and inflammation. Immunesystems with a predominance of inhibitory signals, such as thecostimulatory signaling between CTLA4 and are less able to challengeinfected cells or cancer cells. Isolating new molecules involved in Tcell signaling is highly desirable for studying the biological signal(s)transduced via their receptors. Additionally, identifying such moleculesprovides a means of regulating and treating diseased states associatedwith autoimmunity, inflammation and infection.

SUMMARY OF THE INVENTION

The present invention provides mammalian polypeptides, designated LDCAM,so designated because they are found on lymphoid derived dendritic cellsand display a limited homology to adhesion molecules, including B7-1.The LDCAM molecules described herein include isolated or homogeneousproteins that bind to themselves, have limited homology with B7L-1(described in copending application Ser. No. 60/095,663 filed Aug. 7,1998 (incorporated herein by reference) and for which B7-L1 is a bindingprotein. The present invention further includes isolated DNAs encodingLDCAM and expression vectors comprising DNA encoding mammalian LDCAM.Within the scope of this invention are host cells that have beentransfected or transformed with expression vectors that comprise a DNAencoding LDCAM, and processes for producing LDCAM by culturing such hostcells under conditions conducive to expression of LDCAM. Further withinthe present invention are pharmaceutical compositions comprising solubleforms of LDCAM molecules and methods for modulating T cell immuneresponses by administering the pharmaceutical compositions. Additionalmethods encompassed by the present invention include generating naturalkiller cells by administering pharmaceutical compositions to anindividuals or by combining LDCAM and natural killer cell precursorcells ex vivo.

DETAILED DESCRIPTION OF THE INVENTION

Novel proteins designated LDCAM are provided herein. Further providedare DNA encoding LDCAM, recombinant expression vectors comprising LDCAM,and methods for producing recombinant LDCAM polypeptides that includecultivating host cells transformed with an expression vector underconditions appropriate for expressing LDCAM and recovering the expressedLDCAM.

B7L-1, a molecule having sequence similarity to B7-1, described incopending application Ser. No. 60/095,663 filed Aug. 7, 1998, is abinding protein for the LDCAM polypeptides of the present invention.Because B7L-1 is a LDCAM binding protein and because B7L-1 and LDCAMdisplay homology within their intracellular domain that includespotential binding sites for band 4.1 and PDZ family members, and arefound on many of the same cell types, their cell bound forms may deliversimilar signals when engaged. Thus, they are termed co-receptors orcounterstructures. The nucleotide sequence encoding long and shortextracellular forms of human B7L-1 are presented in SEQ ID NO:7 and SEQID NO:9, respectively. The amino acid sequences encoded by thenucleotide sequences of SEQ ID NO:7 and SEQ ID NO:9 are disclosed in SEQID NO:8 and SEQ ID NO:10, respectively.

To identify cell lines to which B7L-1 binds and to subsequently isolatea protein to which B7L-1 binds, a B7L-1/Fc fusion protein was preparedas described in Example 1 and binding studies, described in Example 2,were carried out. Example 3 describes screening a cDNA library preparedfrom WI-26, a cell line to which B7L-1 binds, and identifying a fulllength LDCAM human clone. The nucleotide sequence encoding human LDCAM,isolated as described in Example 3, is presented in SEQ ID NO:1, and theamino acid sequence encoded thereby is presented in SEQ ID NO:2. Theencoded human LDCAM amino acid sequence described in SEQ ID NO:2 has apredicted extracellular domain of 374 amino acids including a leadersequence of 38 amino acids 1-38; a transmembrane domain of 21 aminoacids (375-395) and a cytoplasmic domain of 47 amino acids (396-442).

Examples 5 and 6 describe making and using a human LDCAM/Fc in bindingstudies to identify cell lines to which the human LDCAM binds. Amongcell lines positively identified were S49.1 cells and lymphoid dendriticcells from spleens and lymph nodes of Flt3-L treated mice. Example 7describes screening pools of an expression library to identify murineLDCAM clones. The isolated murine LDCAM DNA sequence is disclosed in SEQID NO:3. The amino acid sequence encoded by the nucleotide sequence ofSEQ ID NO:3 is disclosed in SEQ ID NO:4. The encoded murine LDCAM aminoacid sequence (SEQ ID NO:4) has a predicted extracellular domain of 356amino acids (residues 1-356); a transmembrane domain of 21 amino acids(357-377); and a cytoplasmic domain that includes amino acid residues378-423. SEQ ID NO:3 and SEQ ID NO:4 describes the full length maturemurine LDCAM sequences. As compared to the human LDCAM sequence, thesignal sequence is not completely described.

The purified mammalian LDCAM molecules described herein are Type Itransmembrane proteins having limited overall homology to B7-1 and othercell adhesion molecules. LDCAM has high homology to the cytoplasmicregion of B7L-1. As described below in Example 6, LDCAM proteinsdemonstrate widespread expression. In particular, human LDCAM mRNA isfound in breast, retina, fetal liver spleen, fetal heart, lung, muscle,placenta, thyroid, and lung carcinoma. Cell lines that have LDCAMmessage include Wi-26. Mouse LDCAM mRNA is found on whole embryo,testes, triple negative cells murine splenic and lymph node CD8⁺, S49.1and dendritic cells.

The discovery of the DNA sequences disclosed in SEQ ID NOs:1 and 3enables construction of expression vectors comprising DNAs encodinghuman and mouse LDCAM proteins; host cells transfected or transformedwith the expression vectors; biologically active LDCAM as homogeneousproteins; and antibodies immunoreactive with LDCAM.

Like B7L-1, LDCAM has limited homology to poliovirus receptor, deltaopoid binding protein and adhesion molecules. Moreover, as described inExample 13, LDCAM blocks T cell proliferation caused by ConA and PHA,suggesting the LDAM is useful in modulating T cell mediated immuneresponse. LDCAM does not inhibit TCR mAb induced T cell proliferationsuggesting that the inhibitory effects of LDCAM on mitogen-induced Tcell proliferation is due to inhibition of cytokine secretion, e.g.IL-2, or due to the regulation of downstream responses of the T cellfollowing activation and increases in the expression of the LDCAMbinding partner. While not limited to such, particular uses of the LDCAMmolecules are described infra.

As used herein, the term LDCAM encompasses polypeptides having the aminoacid sequence 1-442 of SEQ ID NO:2 and the amino acid sequence 1-423 ofSEQ ID NO:4. In addition, LDCAM encompasses polypeptides that have ahigh degree of similarity or a high degree of identity with the aminoacid sequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:4,and which polypeptides are biologically active. The term “LDCAM” refersto a genus of polypeptides that bind and complex with themselves,polypeptides for which B7L-1 is a binding protein, and polypeptides thatalter T cell signals in response to antigen and mitogens

The term “murine LDCAM” refers to biologically active gene products ofthe DNA of SEQ ID NO:3 and the term “human LDCAM” refers to biologicallyactive gene products of the DNA of SEQ ID NO:1. Further encompassed bythe term “LDCAM” are soluble or truncated proteins that compriseprimarily the B7L-1 co-binding portion of the protein, retain biologicalactivity and are capable of being secreted. Specific examples of suchsoluble proteins are those comprising the sequence of amino acids 1-374of SEQ ID NO:2 and those comprising the sequence of amino acids 1-356 ofSEQ ID NO:4. Alternatively, such soluble proteins can exclude a leadersequence and thus encompass amino acids 39-374 of SEQ ID NO:2.

The term “biologically active” as it refers to LDCAM, means that theLDCAM is capable of altering T cell signals in response to mitogens.

“Isolated” means that LDCAM is free of association with other proteinsor polypeptides, for example, as a purification product of recombinanthost cell culture or as a purified extract.

A “LDCAM variant” as referred to herein, means a polypeptidesubstantially homologous to native LDCAM, but which has an amino acidsequence different from that of native LDCAM (human, murine or othermammalian species) because of one or more deletions, insertions orsubstitutions. The variant amino acid sequence preferably is at least80% identical to a native LDCAM amino acid sequence, most preferably atleast 90% identical. The percent identity may be determined, forexample, by comparing sequence information using the GAP computerprogram, version 6.0 described by Devereux et al. (Nucl. Acids Res.12:387, 1984) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps. Variants may comprise conservatively substituted sequences,meaning that a given amino acid residue is replaced by a residue havingsimilar physiochemical characteristics. Examples of conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another, or substitutions of onepolar residue for another, such as between Lys and Arg; Glu and Asp; orGln and Asn. Other such conservative substitutions, for example,substitutions of entire regions having similar hydrophobicitycharacteristics, are well known. Naturally occurring LDCAM variants oralleles are also encompassed by the invention. Examples of such variantsare proteins that result from alternate mRNA splicing events or fromproteolytic cleavage of the LDCAM protein, wherein the LDCAM bindingproperty is retained. Alternate splicing of mRNA may yield a truncatedbut biologically active LDCAM protein, such as a naturally occurringsoluble form of the protein, for example. Variations attributable toproteolysis include, for example, differences in the N- or C-terminiupon expression in different types of host cells, due to proteolyticremoval of one or more terminal amino acids from the LDCAM protein(generally from 1-5 terminal amino acids).

Example 9 describes the construction of a novel LDCAM/Fc fusion proteinthat may be utilized in LDCAM binding studies and studies directed toexamining functional characteristics of the molecule. Other antibody Fcregions may be substituted for the human IgG1 Fc region described in theExample. Other suitable Fc regions are those that can bind with highaffinity to protein A or protein G, or those that include fragments ofthe human or murine IgG1 Fc region, e.g., fragments comprising at leastthe hinge region so that interchain disulfide bonds will form. The LDCAMfusion protein offers the advantage of being easily purified. Inaddition, disulfide bonds form between the Fc regions of two separatefusion protein chains, creating dimers.

As described supra, an aspect of the invention is soluble LDCAMpolypeptides. Soluble LDCAM polypeptides comprise all or part of theextracellular domain of a native LDCAM but lack the signal that wouldcause retention of the polypeptide on a cell membrane. Soluble LDCAMpolypeptides advantageously comprise the native (or a heterologous)signal peptide when initially synthesized to promote secretion, but thesignal peptide is cleaved upon secretion of LDCAM from the cell. SolubleLDCAM polypeptides encompassed by the invention retain the ability tobind B7L-1, or the ability to bind to themselves. Alternatively solubleLDCAM polypeptides of the present invention retain the ability to alterT cell responses. Soluble LDCAM may include part of the signal or partof the cytoplasmic domain or other sequences, provided that the solubleLDCAM protein can be secreted.

Soluble LDCAM may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired protein from the culture medium, e.g., by centrifugation,and assaying the medium or supernatant for the presence of the desiredprotein. The presence of LDCAM in the medium indicates that the proteinwas secreted from the cells and thus is a soluble form of the desiredprotein.

Soluble forms of LDCAM possess many advantages over the native boundLDCAM protein. Purification of the proteins from recombinant host cellsis feasible, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

Examples of soluble LDCAM polypeptides include those comprising asubstantial portion of the extracellular domain of a native LDCAMprotein. For example, a soluble human LDCAM protein comprises aminoacids 38-374 or 1-374 of SEQ ID NO:2 and a soluble murine LDCAM includesamino acids 1-356 of SEQ ID NO:4. In addition, truncated soluble LDCAMproteins comprising less than the entire extracellular domain areincluded in the invention. When initially expressed within a host cell,soluble LDCAM may include one of the heterologous signal peptidesdescribed below that is functional within the host cells employed.Alternatively, the protein may comprise the native signal peptide. Inone embodiment of the invention, soluble LDCAM can be expressed as afusion protein comprising (from N- to C-terminus) the yeast α-factorsignal peptide, a FLAG® peptide described below and in U.S. Pat. No.5,011,912, and soluble LDCAM consisting of amino acids 39-374 of SEQ IDNO:2 or 21-356 of SEQ ID NO:4. This recombinant fusion protein isexpressed in and secreted from yeast cells. The FLAG® peptidefacilitates purification of the protein, and subsequently may be cleavedfrom the soluble LDCAM using bovine mucosal enterokinase. Isolated DNAsequences encoding soluble LDCAM proteins are encompassed by theinvention.

Truncated LDCAM, including soluble polypeptides, may be prepared by anyof a number of conventional techniques. A desired DNA sequence may bechemically synthesized using techniques known per se. DNA fragments alsomay be produced by restriction endonuclease digestion of a full lengthcloned DNA sequence, and isolated by electrophoresis on agarose gels.Linkers containing restriction endonuclease cleavage site(s) may beemployed to insert the desired DNA fragment into an expression vector,or the fragment may be digested at cleavage sites naturally presenttherein. The well known polymerase chain reaction procedure also may beemployed to amplify a DNA sequence encoding a desired protein fragment.As a further alternative, known mutagenesis techniques may be employedto insert a stop codon at a desired point, e.g., immediately downstreamof the codon for the last amino acid of the receptor-binding domain.

As stated above, the invention provides isolated or homogeneous LDCAMpolypeptides, both recombinant and non-recombinant. Additionally withinthe scope of the present invention are variants and derivatives ofnative LDCAM proteins that retain the desired biological activity. Suchactivity includes the ability of LDCAM to bind to itself, or the abilityto bind to B7L-1, or the ability to alter T cell signaling. LDCAMvariants and derivatives may be obtained by mutations of nucleotidesequences coding for native LDCAM polypeptides. Alterations of thenative amino acid sequence may be accomplished by any of a number ofconventional methods. Mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462all of which are incorporated by reference.

LDCAM may be modified to create LDCAM derivatives by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of LDCAM may be prepared by linking the chemical moieties tofunctional groups on LDCAM amino acid side chains or at the N-terminusor C-terminus of a LDCAM polypeptide or the extracellular domainthereof. Other derivatives of LDCAM within the scope of this inventioninclude covalent or aggregative conjugates of LDCAM or its fragmentswith other proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatemay comprise a signal or leader polypeptide sequence (e.g. the α-factorleader of Saccharomyces) at the N-terminus of a LDCAM polypeptide. Thesignal or leader peptide co-translationally or post-translationallydirects transfer of the conjugate from its site of synthesis to a siteinside or outside of the cell membrane or cell wall.

LDCAM polypeptide fusions can comprise peptides added to facilitatepurification and identification of LDCAM. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.

The invention further includes LDCAM polypeptides with or withoutassociated native-pattern glycosylation. LDCAM expressed in yeast ormammalian expression systems (e.g., COS-7 cells) may be similar to orsignificantly different from a native LDCAM polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of LDCAM polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity orbinding are encompassed by the invention. For example, N-glycosylationsites in the LDCAM extracellular domain can be modified to precludeglycosylation, allowing expression of a reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Thehuman LDCAM polypeptide of SEQ ID NO:2 includes six such triplets, atamino acids 67-69, 101-103, 113-115, 165-167, 304-306, and 308-310.Similarly, the murine LDCAM polypeptide of SEQ ID NO:4 includes sic suchtriplets at 49-51, 83-85, 95-97, 147-149, 286-288 and 290-292.Appropriate substitutions, additions or deletions to the nucleotidesequence encoding these triplets will result in prevention of attachmentof carbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid,for example, is sufficient to inactivate an N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the LDCAM nucleotidesequences disclosed herein under conditions of moderate or severestringency, and that encode biologically active LDCAM. Conditions ofmoderate stringency, as defined by Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold Spring HarborLaboratory Press, (1989), include use of a prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions ofabout 55° C., 5 ×SSC, overnight. Conditions of severe stringency includehigher temperatures of hybridization and washing. The skilled artisanwill recognize that the temperature and wash solution salt concentrationmay be adjusted as necessary according to factors such as the length ofthe nucleic acid molecule.

Due to the known degeneracy of the genetic code wherein more than onecodon can encode the same amino acid, a DNA sequence may vary from thatshown in SEQ ID NO:1 and 3 and still encode a LDCAM protein having theamino acid sequence of SEQ ID NO:2 and SEQ ID NO:4, respectively. Suchvariant DNA sequences may result from silent mutations (e.g., occurringduring PCR amplification), or may be the product of deliberatemutagenesis of a native sequence.

The invention provides equivalent isolated DNA sequences encodingbiologically active LDCAM, selected from: (a) cDNA comprising thenucleotide sequence presented in SEQ ID NO:1 and SEQ ID NO:3; (b) DNAcapable of hybridization to a DNA of (a) under moderately stringentconditions and that encodes biologically active LDCAM; and (c) DNA thatis degenerate as a result of the genetic code to a DNA defined in (a),or (b) and that encodes biologically active LDCAM. LDCAM proteinsencoded by such DNA equivalent sequences are encompassed by theinvention.

DNAs that are equivalent to the DNA sequence of SEQ ID NO:1 and SEQ IDNO:3 will hybridize under moderately and severely stringent conditionsto DNA sequences that encode polypeptides comprising SEQ ID NO:2, andSEQ ID NO:4. Examples of LDCAM proteins encoded by such DNA, include,but are not limited to, LDCAM fragments (including soluble fragments)and LDCAM proteins comprising inactivated N-glycosylation site(s),inactivated KEX2 protease processing site(s), or conservative amino acidsubstitution(s), as described above. LDCAM proteins encoded by DNAderived from other mammalian species, wherein the DNA will hybridize tothe cDNA of SEQ ID NO:1 or SEQ ID NO:3 are also encompassed by thepresent invention.

Variants possessing the ability to bind B7L-1 may be identified by anysuitable assay. Biological activity of LDCAM may be determined, forexample, by competition for binding to the binding domain of B7L-1 (i.e.competitive binding assays).

One type of a competitive binding assay for a LDCAM polypeptide uses aradiolabeled, soluble LDCAM and intact cells expressingB7L-1-expressing. Instead of intact cells, one could substitute solubleB7L-1/Fc fusion proteins such as a B7L-1/Fc bound to a solid phasethrough the interaction of a Protein A, Protein G or an antibody to theB7L-1 or Fc portions of the molecule, with the Fc region of the fusionprotein. Another type of competitive binding assay utilizes aradiolabeled soluble LDCAM receptor and intact cells expressing LDCAM.

Competitive binding assays can be performed following conventionalmethodology. For example, radiolabeled LDCAM can be used to compete witha putative LDCAM homologue to assay for binding activity against B7L-1or a surface-bound LDCAM receptor. Qualitative results can be obtainedby competitive autoradiographic plate binding assays, or Scatchard plotsmay be utilized to generate quantitative results.

Alternatively, LDCAM-binding proteins, such as B7L-1 and anti-LDCAMantibodies, can be bound to a solid phase such as a columnchromatography matrix or a similar substrate suitable for identifying,separating or purifying cells that express LDCAM on their surface.Binding of a LDCAM-binding protein to a solid phase contacting surfacecan be accomplished by any means, for example, by constructing aB7L-1/Fc fusion protein and binding such to the solid phase through theinteraction of Protein A or Protein G. Various other means for fixingproteins to a solid phase are well known in the art and are suitable foruse in the present invention. For example, magnetic microspheres can becoated with B7L-1 and held in the incubation vessel through a magneticfield. Suspensions of cell mixtures containing LDCAM-expressing cellsare contacted with the solid phase that has B7L-1 polypeptides thereon.Cells having LDCAM on their surface bind to the fixed B7L-1 and unboundcells then are washed away. This affinity-binding method is useful forpurifying, screening or separating such LDCAM-expressing cells fromsolution. Methods of releasing positively selected cells from the solidphase are known in the art and encompass, for example, the use ofenzymes. Such enzymes are preferably non-toxic and non-injurious to thecells and are preferably directed to cleaving the cell-surface bindingpartner. In the case of B7L-1:LDCAM interactions, the enzyme preferablyfrees the resulting cell suspension from the LDCAM material. Thepurified cell population, especially if obtained from fetal tissue, thenmay be used to repopulate mature (adult) tissues.

Alternatively, mixtures of cells suspected of containing LDCAM⁺ cellsfirst can be incubated with biotinylated B7L-1. Incubation periods aretypically at least one hour in duration to ensure sufficient binding toLDCAM The resulting mixture then is passed through a column packed withavidin-coated beads, whereby the high affinity of biotin for avidinprovides the binding of the cell to the beads. Use of avidin-coatedbeads is known in the art. See Berenson, et al. J. Cell. Biochem.,10D:239 (1986). Wash of unbound material and the release of the boundcells is performed using conventional methods.

As described above, B7L-1 can be used to separate cells expressingLDCAM, and,. In an alternative method, LDCAM or an extracellular domainor a fragment thereof can be conjugated to a detectable moiety such as1251 to detect B7L-1-expressing cells. Radiolabeling with ¹²⁵I can beperformed by any of several standard methodologies that yield afunctional ¹²⁵I-LDCAM molecule labeled to high specific activity. Or aniodinated or biotinylated antibody against the B7L-1 region or the Fcregion of the molecule could be used. Another detectable moiety such asan enzyme that can catalyze a colorimetric or fluorometric reaction,biotin or avidin may be used. Cells to be tested for B7L-1-expressioncan be contacted with labeled LDCAM. After incubation, unbound labeledLDCAM is removed and binding is measured using the detectable moiety.

The binding characteristics of LDCAM (including variants) may also bedetermined using the conjugated, soluble LDCAM/Fc (for example,¹²⁵I-LDCAM/Fc) in competition assays similar to those described above.In this case, however, intact cells expressing LDCAM/Fc bound to a solidsubstrate, are used to measure the extent to which a sample containing aputative LDCAM variant competes for binding with a conjugated solublebinding partner for LDCAM.

Other means of assaying for LDCAM include the use of anti-LDCAMantibodies, cell lines that proliferate in response to LDCAM, orrecombinant cell lines that proliferate in the presence of LDCAM.

The LDCAM proteins disclosed herein also may be employed to measure thebiological activity of B7L-1 or other LDCAM binding proteins in terms oftheir binding affinity for LDCAM. As one example, LDCAM may be used indetermining whether biological activity is retained after modificationof B7L-1 (e.g., chemical modification, truncation, mutation, etc.). Thebiological activity of a B7L-1 protein thus can be ascertained before itis used in a research study, or possibly in the clinic, for example.

LDCAM proteins find use as reagents that may be employed by thoseconducting “quality assurance” studies, e.g., to monitor shelf life andstability of B7L-1 or other LDCAM binding protein under differentconditions. To illustrate, LDCAM may be employed in a binding affinitystudy to measure the biological activity of an B7L-1 protein that hasbeen stored at different temperatures, or produced in different celltypes. The binding affinity of the modified B7L-1 protein for LDCAM iscompared to that of an unmodified B7L-1 protein to detect any adverseimpact of the modifications on biological activity of B7L-1. Likewise,the biological activity of a LDCAM protein can be assessed using B7L-1.

LDCAM polypeptides also find use as carriers for delivering agentsattached thereto to T cells or other cells bearing B7L-1 or LDCAM. LDCAMproteins can be used to deliver diagnostic or therapeutic agents tothese cells in in vitro or in vivo procedures. As described in Example5, LDCAM is found on the PAE81BM cell line, which is an EBV transformedcell line. Thus, one example of such carrier use is to expose this cellline to a therapeutic agent/LDCAM conjugate to assess whether the agentexhibits cytotoxicity toward any EBV cancers. Additionally, since LDCAMis expressed on dendritic cells and CD40L activated B cells that areimportant in antigen presentation, LDCAM is a useful carrier fortargeting, identifying, and purifying these cells. Also,LDCAM/diagnostic agent conjugates may be employed to detect the presenceof dendritic cells and B cells in vitro or in vivo. Example 6demonstrates that human LDCAM mRNA, transcripts are found in humanbreast, retinal, fetal liver, spleen, fetal heart, lung, placenta,thyroid and lung carcinoma. Similar studies for expression of mouseLDCAM mRNA showed that mouse LDCAM mRNA is found in whole embryo,testes, lymphoid derived dendritic cells and triple negative cells.Since, LDCAM binds to itself, LDCAM can be used to study its functionalrole in these tissues.

A number of different therapeutic agents or other functional markersattached to LDCAM may be used in conjugates in an assay to detect andcompare the cytotoxic effects of the agents on the cells or study therole of LDCAM in tissues and cells. Diagnostic and therapeutic agentsthat may be attached to a LDCAM polypeptide include, but are not limitedto, drugs, toxins, radionuclides, chromophores, enzymes that catalyze acolorimetric or fluorometric reaction, and the like, with the particularagent being chosen according to the intended application. Examples ofdrugs include those used in treating various forms of cancer, e.g.,nitrogen mustards such as L-phenylalanine nitrogen mustard orcyclophosphamide, intercalating agents such ascis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil,vinca alkaloids such as vincristine, and antibiotics such as bleomycin,doxorubicin, daunorubicin, and derivatives thereof. Among the toxins arericin, abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A,ribosomal inactivating proteins, mycotoxins such as trichothecenes, andderivatives and fragments (e.g., single chains) thereof. Radionuclidessuitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I,⁹⁹mTc, ¹¹¹In, and ⁷⁶Br. Radionuclides suitable for therapeutic useinclude, but are not limited to, ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb,²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

Such agents may be attached to the LDCAM by any suitable conventionalprocedure. LDCAM, being a protein, comprises functional groups on aminoacid side chains that can be reacted with functional groups on a desiredagent to form covalent bonds, for example. Alternatively, the protein oragent may be derivatized to generate or attach a desired reactivefunctional group. The derivatization may involve attachment of one ofthe bifunctional coupling reagents available for attaching variousmolecules to proteins (Pierce Chemical Company, Rockford, Ill.). Anumber of techniques for radiolabeling proteins are known. Radionuclidemetals may be attached to LDCAM by using a suitable bifunctionalchelating agent, for example.

Conjugates comprising LDCAM and a suitable diagnostic or therapeuticagent (preferably covalently linked) are thus prepared. The conjugatesare administered or otherwise employed in an amount appropriate for theparticular application.

As mentioned above, because LDCAM blocks T cell proliferation caused byConA and PHA and does not inhibit TCR mAb induced T cell proliferation,the inhibitory effects of LDCAM on mitogen-induced T cell proliferationis likely due to the inhibition of cytokine secretion, e.g. IL-2.Accordingly, another use of the LDCAM of the present invention is as aresearch tool for studying the role that LDCAM plays in the productionof IL-2 in T cells. The LDCAM polypeptides of the present invention alsomay be employed in in vitro assays for detection of B7L-1 or theinteractions thereof.

One embodiment of the present invention is directed to a method oftreating disorders associated with a malfunctioning immune system. Moreparticularly, since LDCAM is known to block ConA stimulated T cells andPHA stimulated T cells, LDCAM may be useful in treating inflammation andautoimmune disorders mediated by T cell responses. A composition thatincludes a LDCAM protein, preferably a soluble polypeptide, and apharmaceutically acceptable diluent or carrier may be administered to amammal to treat such inflammation or autoimmune disorder.

SCID mice that have been injected with soluble LDCAM, in the form ofLDCAM/Fc, experience an increase in splenic cellularity. Part of thisincrease is due to an increase in DX-5⁺ cells, also known as naturalkiller cells (NK cells). When injected with LDCAM/Fc and IL-15, a NKcell growth factor, SCID mice demonstrate an increase in NK cells thatis additive. This further evidences the ability of LDCAM, LDCAMfragments, and soluble LDCAM to generate NK cells. In view of thisdiscovery, another embodiment of the present invention includes methodsfor increasing the number of NK cells in an individual by administering,to that individual, pharmaceutical compositions, of the presentinvention, containing LDCAM, soluble LDCAM, or LDCAM fragments. Inanother embodiment, NK cells may be increased ex vivo by contacting NKcells with LDCAM or soluble forms of LDCAM and allowing the NK cells toexpand. Similarly, NK cells can be generated in vivo or ex vivo, as justdescribed, by administering LDCAM or soluble forms of LDCAM inconnection with additional cytokines or growth factors. Thus, thepresent methods for generating NK cells, in vivo or ex vivo can furtherinclude the use of an effective amount of a cytokine in sequential orconcurrent combination with LDCAM. Such cytokines include, but are notlimited to, interleukins (“ILs”) IL-15, IL-3 and IL-4, a colonystimulating factor (“CSF”) selected from the group consisting ofgranulocyte macrophage colony stimulating factor (“GM-CSF”) orGM-CSF/IL-3 fusions, or other cytokines such as TNF-α, CD40 bindingproteins (e.g. CD40-L), 4-1 BB antagonists (e.g. antibodiesimmunoreactive with 4-1BB and 4-1 BB-L) or c-kit ligand.

NK cells are large granular lymphocytes that are distinct from T or Blymphocytes in morphology and function. NK cells mediate killing certaintumor cells and virally infected cells in non-MHC restricted manners.Additionally, NK cells are involved in the rejection of donor cells bybone marrow transplant recipients. Since LDCAM increases NK cellnumbers, LDCAM, soluble LDCAM, or LDCAM fragments are useful incombating virally infected cells and infectious diseases. Similarly,LDCAM, soluble LDCAM, and LDCAM fragments are useful for killing tumorcells. Accordingly, within the scope of the present invention aremethods for treating infectious diseases and methods for treatingindividuals afflicted with tumors. Such therapeutic methods involveadministering LDCAM, soluble forms of LDCAM, or LDCAM fragments to anindividual in need of increasing their numbers of NK cells in order tokill tumor cells or enhance their ability to combat infectious disease.Similarly, the therapeutic methods of the present invention can becarried out by administering LDCAM, soluble LDCAM, e.g. LDCAM fusionprotein, or LDCAM fragments sequentially or concurrently in combinationcytokines. Such cytokines include, but are not limited to, interleukins(“ILs”) IL-15, IL-3 and IL-4, a colony stimulating factor (“CSF”)selected from the group consisting of granulocyte macrophage colonystimulating factor (“GM-CSF”) or GM-CSF/IL-3 fusions, or other cytokinessuch as TNF-α, CD40 binding proteins (e.g. CD40-L), 4-1BB antagonists(e.g. antibodies immunoreactive with 4-1BB and 4-1 BB-L) or c-kitligand.

Further within the scope of the present invention are methods forpreventing or decreasing the effect of organ and bone marrow transplantrejection by recipients of the transplant. Such methods involve treatingrecipients with a composition that includes a LDCAM inhibitor, thusinhibiting increases in NK cell populations and decreasing the abilityof NK cells to reject transplants.

Treatment of human endothelial cells (aortic and umbilical cord) with asoluble form of human LDCAM results in calcium fluxes within the cells.Calcium fluxes in endothelial cells are important in modulating vascularpermeability, endothelial cell migration and angiogenesis, and adhesionand transmigration of leukocytes. LDCAM polypeptides and LDCAMinhibitors may therefore be used to improve drug delivery across theblood-brain barrier, to augment an immune response against a tumor orpathogen, to lessen an autoimmune or inflammatory syndrome, to lessenleukocyte adhesion and formation of atherosclerotic plaques, to blockangiogenesis, and in the treatment of pathogenic vascular leakage.

LDCAM polypeptides of the invention can be formulated according to knownmethods used to prepare pharmaceutically useful compositions. LDCAM canbe combined in admixture, either as the sole active material or withother known active materials, with pharmaceutically suitable diluents(e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co.In addition, such compositions can contain LDCAM complexed withpolyethylene glycol (PEG), metal ions, or incorporated into polymericcompounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.,or incorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance of LDCAM. LDCAMcan also be conjugated to antibodies against tissue-specific receptors,ligands or antigens, or coupled to ligands of tissue-specific receptors.Where a LDCAM binding protein is found on tumor cells, the LDCAM may beconjugated to a toxin whereby LDCAM is used to deliver the toxin to thespecific site, or may be used to sensitize such tumor cells tosubsequently administered agents.

LDCAM can be administered topically, parenterally, or by inhalation. Theterm “parenteral” includes subcutaneous injections, intravenous,intramuscular, intracistemal injection, or infusion techniques. Thesecompositions will typically contain an effective amount of the LDCAM,alone or in combination with an effective amount of any other activematerial. Such dosages and desired drug concentrations contained in thecompositions may vary depending upon many factors, including theintended use, patient's body weight and age, and route ofadministration. Preliminary doses can be determined according to animaltests, and the scaling of dosages for human administration can beperformed according to art-accepted practices.

LDCAM polypeptides may exist as oligomers, such as covalently linked ornon-covalently-linked dimers or trimers. Oligomers may be linked bydisulfide bonds formed between cysteine residues on different LDCAMpolypeptides. In one embodiment of the invention, a LDCAM dimer iscreated by fusing LDCAM to the Fc region of an antibody (e.g., IgG1) ina manner that does not interfere with binding of LDCAM to the T cells,B7L-1 or itself. The Fc polypeptide preferably is fused to theC-terminus of a soluble LDCAM (comprising only the receptor binding).General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the LDCAM:Fc fusionprotein is inserted into an appropriate expression vector. LDCAM:Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding divalent LDCAM. If fusion proteins are made with both heavy andlight chains of an antibody, it is possible to form a LDCAM oligomerwith as many as four LDCAM extracellular regions. Alternatively, one canlink two soluble LDCAM domains with a peptide linker.

Suitable host cells for expression of LDCAM polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce LDCAM polypeptidesusing RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a LDCAM polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant LDCAM polypeptide.

LDCAM polypeptides may be expressed in yeast host cells, preferably fromthe Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast,such as Pichia , K. lactis or Kluyveromyces, may also be employed. Yeastvectors will often contain an origin of replication sequence from a 2μyeast plasmid, an autonomously replicating sequence (ARS), a promoterregion, sequences for polyadenylation, sequences for transcriptiontermination, and a selectable marker gene. Suitable promoter sequencesfor yeast vectors include, among others, promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van denBerg et. al., Bio/Technology, 8:135-139 (1990). Another alternative isthe glucose-repressible ADH2 promoter described by Russell et al. (J.Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982).Shuttle vectors replicable in both yeast and E. coli may be constructedby inserting DNA sequences from pBR322 for selection and replication inE. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the LDCAM polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad.Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Otherleader sequences suitable for facilitating secretion of recombinantpolypeptides from yeast hosts are known to those of skill in the art. Aleader sequence may be modified near its 3′ end to contain one or morerestriction sites. This will facilitate fusion of the leader sequence tothe structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant LDCAM polypeptides. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Exemplary expression vectors for use in mammalian host cells can beconstructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses. In place of the native signal sequence, andin addition to an initiator methionine, a heterologous signal sequencemay be added, such as the signal sequence for IL-7 described in U.S.Pat. No. 4,965,195; the signal sequence for IL-2 receptor described inCosman et al., Nature 312:768 (1984); the IL-4 signal peptide describedin EP 367,566; the type I IL-1 receptor signal peptide described in U.S.Pat. No. 4,968,607; and the type II IL-1 receptor signal peptidedescribed in EP 460,846.

LDCAM as an isolated, purified or homogeneous protein according to theinvention may be produced by recombinant expression systems as describedabove or purified from naturally occurring cells. LDCAM can be purifiedto substantial homogeneity, as indicated by a single protein band uponanalysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

One process for producing LDCAM comprises culturing a host celltransformed with an expression vector comprising a DNA sequence thatencodes LDCAM under conditions sufficient to promote expression ofLDCAM. LDCAM is then recovered from culture medium or cell extracts,depending upon the expression system employed. As is known to theskilled artisan, procedures for purifying a recombinant protein willvary according to such factors as the type of host cells employed andwhether or not the recombinant protein is secreted into the culturemedium.

For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify LDCAM. Some or all of the foregoingpurification steps, in various combinations, are well known and can beemployed to provide a substantially homogeneous recombinant protein.

It is possible to utilize an affinity column comprising theligand-binding domain of a LDCAM binding protein to affinity-purifyexpressed LDCAM polypeptides. LDCAM polypeptides can be removed from anaffinity column using conventional techniques, e.g., in a high saltelution buffer and then dialyzed into a lower salt buffer for use or bychanging pH or other components depending on the affinity matrixutilized. Alternatively, the affinity column may comprise an antibodythat binds LDCAM. Example 10 describes a procedure for employing LDCAMof the invention to generate monoclonal antibodies directed againstLDCAM.

Recombinant protein produced in bacterial culture can be isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express LDCAM asa secreted polypeptide in order to simplify purification. Secretedrecombinant polypeptide from a yeast host cell fermentation can bepurified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

Useful fragments of the LDCAM nucleic acids include antisense or senseoligonucleotides comprising a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target LDCAM mRNA (sense) orLDCAM DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of LDCAM cDNA. Such a fragment generally comprises at least about14 nucleotides, preferably from about 14 to about 30 nucleotides. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein is described in, for example,Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of LDCAM proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increases affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US90/02656).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a sense or an antisenseoligonucleotide may be introduced into a cell containing the targetnucleic acid sequence by formation of an oligonucleotide-lipid complex,as described in WO 90/10448. The sense or antisenseoligonucleotide-lipid complex is preferably dissociated within the cellby an endogenous lipase.

In addition to the above, the following examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Preparing B7L-1/Fc Fusion Protein

The following describes generating a human B7L-1/Fc protein which wasused to identify cells to which B7L-1 binds. The fusion protein includesthe soluble extracellular region of human B7L-1 and the mutein human Fcregion and was prepared by first isolating cDNA encoding theextracellular region of human B7L-1 using primers which flank theextracellular region of B7L-1 (See U.S. Pat. No. 5,011,912).

To isolate the nucleotides that encode the extracellular domain of B7L-1(nucleotides 108-1249 of SEQ ID NO:1 of copending application Ser. No.60/095,663 filed Aug. 7, 1998 ) oligonucleotides that flank theextracellular region of B7L-1 were used as primers in a PCR reaction toobtain a PCR product from clone #44904 which was the template in thereaction. The resulting PCR product was digested with Sal1 and Bg1IIrestriction enzymes at the Sal1 and Bg1II sites incorporated by theprimers. The resulting fragment was ligated into an expression vector(pDC409) containing the human IgG1 Fc region mutated to lower Fcreceptor binding.

The resulting DNA construct was transfected into the monkey kidney celllines CV-1/EBNA (with co-transfection of psv3neo). After 7 days ofculture in medium containing 0.5% low immunoglobulin bovine serum, asolution of 0.2% azide was added to the supernatant and the supernatantwas filtered through a 0.22 μm filter. Then approximately 1 L of culturesupernatant was passed through a BioCad Protein A HPLC proteinpurification system using a 4.6×100 mm Protein A column (POROS 20A fromPerSeptive Biosystems) at 10 mL/min. The Protein A column binds the FcPortion of the fusion protein in the supernatant, immobilizing thefusion protein and allowing other components of the supernatant to passthrough the column. The column was washed with 30 mL of PBS solution andbound fusion protein was eluted from the HPLC column with citric acidadjusted to pH 3.0. Eluted purified fusion protein was neutralized as iteluted using 1M HEPES solution at pH 7.4.

EXAMPLE 2 B7L-1 Binding Studies

The B7L-1/Fc fusion protein prepared as described in Example 1 was usedto screen cell lines for B7L-1 binding using quantitative bindingstudies according to standard flow cytometry methodologies. For eachcell line screened, the procedure involved incubating cells blocked with2% FCS (fetal calf serum), 5% normal goat serum and 5% rabbit serum inPBS for 1 hour. Then the blocked cells were incubated with 5 μg/mL ofB7L-1/Fc fusion protein in 2% FCS, 5% goat serum and 5% rabbit serum inPBS. Following the incubation the sample was washed 2 times with FACSbuffer (2% FCS in PBS) and then treated with mouse anti human Fc/biotin(purchased from Jackson Research) and SAPE (streptavidin-phycoerythrinpurchased from Molecular Probes). This treatment causes the antihumanFc/biotin to bind to any bound B7L-1/Fc and the SAPE to bind to theanti-human Fc/biotin resulting in a fluorescent identifying label onB7L-1/Fc which is bound to cells. The cells were analyzed for any boundprotein using fluorescent detection flow cytometry. The resultsindicated that human B7L-1 binds well to human lung epithelial line(WI-28), human B lymphoblastoid lines (Daudi and PAE8LBM1), human freshtonsillar B cells, murine CD8⁺ dendritic cells from spleens/lymph nodesof flt3-L treated animals and murine T cell lymphoma S49.1.

EXAMPLE 3 Screening WI-26 Expression Library for B7L-1 Counter Receptors

The following describes screening a expression cloning library with theB7L-1/Fc fusion protein prepared as described in Example 1. Theexpression library was prepared from the human cell line WI-26 usingmethods described in Current Protocols In Molecular Biology, Vol. 1,(1987). Using standard indirect-binding methods, transfected monolayersof CV1/EBNA cells were assayed by slide autoradiography for expressionof a B7L-1 counter receptor using radioiodinated B7L-1/Fc fusionprotein. Positive slides showing cells expressing a counter receptorwere identified and one pool containing approximately 2,000 individualclones was identified as potentially positive for binding the B7L-1/Fcfusion protein.

The pool was titered and plated and then scraped to provide pooledplasmid DNA for transfection into CV1/EBNA cells. After screening thesmaller pools, one pool contained clones that were positive for B7L-1counter receptor as indicated by the presence of an expressed geneproduct capable of binding to B7L-1/Fc. The positive pool was titeredand plated to obtain individual colonies. DNA was isolated from eachpotential candidate clone, retransfected and rescreened. The resultingpositive clones contained a cDNA insert of 1535 nucleotides. The cDNAcoding region of the B7L-1 counter receptor (LDCAM) corresponds to thatdisclosed SEQ ID NO:1. The amino acid sequence encoded by SEQ ID NO:1 isdisclosed in SEQ ID NO:2.

EXAMPLE 4 Expressing Human LDCAM

To following describes expressing full length membrane-bound human LDCAMin CV1/EBNA cells. A vector construct for expressing human LDCAM wasprepared by ligating the coding region of SEQ ID NO:1 into a pDC409expression vector. The expression vector was then transfected inCV1/EBNA cells and LDCAM was expressed using techniques described inMcMahan et al., EMBO J. 10:2821,1991.

After the cells were shocked and incubated for several days, cellshaving membrane bound LDCAM were harvested, fixed in 1%paraformaldehyde, washed and used in their intact form.

To express a soluble form of LDCAM that includes the LDCAM extracellularregion encoded by nucleotides 8 to 1130 of SEQ ID NO:1, a vectorconstruct is prepared by ligating the extracellular coding region of SEQID NO:1 into a pDC409 expression vector. The vector is transfected inCV1/EBNA cells

Following a 3 day incubation period in fresh medium, soluble LDCAM isrecovered by collecting CV1/EBNA cell supernatants containing thesoluble form and isolating LDCAM using HPLC techniques or affinitychromatography techniques.

EXAMPLE 5 LDCAM Binding Studies

In order to identify cell lines to which LDCAM binds, the LDCAM/Fcfusion protein, described in Example 9 below, was prepared and used incell binding and FACS assays. Using standard cell binding and FACSmethodologies, LDCAM was found to bind to the B lymphoblastoid celllines, DAUDI and PAE8LBM1, cells transfected with human B7L-1, cellstransfected with LDCAM, S49.1 cells, and to the lymphoid DCs fromspleens and lymph nodes of Flt3-L treated mice.

EXAMPLE 6 Identifying Tissue Expressing LDCAM

Using standard RT-PCR methodologies, Northern analyses and EST data base(GENBANK) sequence matching, a number of cell lines were examined formRNA expression of human LDCAM and mouse LDCAM. The results demonstratedthat LDCAM has a widespread tissue distribution. Expression of humanLDCAM was found in breast, retina, fetal liver, spleen, fetal heart,lung, muscle, placenta, thyroid, and lung carcinoma. Mouse mRNA LDCAMwas found in whole embryo, testes, and triple negative cells.

EXAMPLE 7 Isolating Murine LDCAM

Since the soluble human B7L-1 demonstrated binding to the murinelymphoma S49.1 (Example 2), a S49.1 expression library was screened formurine LDCAM cDNA clones. The process involved RT-PCR methodologiesusing the S49.1 cell line RNA and primers described in SEQ ID NO:7 andSEQ ID NO:8. These primers are based on a murine EST, discovered in adatabase and having homology to human LDCAM. The cDNAs were amplified byPCR using the primers, confirming the murine LDCAM is present in S49.1cells.

The amplified product was cloned into a cloning vector and clonescontaining a LDCAM cDNA insert were detected by hybridization with anoligonucleotide complementary to the human LDCAM coding region. Todetect cDNAs with 5′ extensions as compared with human LDCAM anoligonucleotide primer complementary to the 5′ end of the coding regionand a primer complementary to vector sequences adjacent to the cDNAinsert were used to perform anchored PCR so that the 5′ region of thecDNA clones is amplified. The PCR products were examined by gelelectrophoresis and their lengths were compared with a similarly derivedamplification product from the human LDCAM cDNA. The cDNA inserts forthe clones giving longer 5′ PCR product were sequenced to give a murineLDCAM cDNA encoding all but the first 4 amino acids, as compared withthe human LDCAM. The nucleotide sequence for murine LDCAM is given inSEQ ID NO:3. The amino acid sequence encoded by the nucleotide sequenceof SEQ ID NO:3 is provided in SEQ ID NO:4.

EXAMPLE 8 Expressing Murine LDCAM Polypeptide

To prepare a vector construct for expressing murine extracellular B7L-1the coding region of SEQ ID NO:3 was ligated into a pDC409 expressionvector. The expression vector was then transfected in CV1/EBNA cells andLDCAM was expressed using techniques described in McMahan et al., EMBOJ. 10:2821,1991.

After the cells were shocked and incubated for several days, cellsupernatants containing soluble murine LDCAM were collected and theprotein was recovered using HPLC techniques.

EXAMPLE 9 Preparing LDCAM/Fusion Proteins

The following describes generating a human LDCAM/Fc protein which wasused to identify cells to which LDCAM binds. The fusion protein includesthe soluble extracellular region of human LDCAM and the mutein human Fcregion and was prepared by first isolating cDNA encoding theextracellular region of human LDCAM using primers which flank theextracellular region of LDCAM (See U.S. Pat. No. 5,011,912).

To isolate the nucleotides that encode the extracellular domain ofLDCAM, nucleotides 16-1137 of SEQ ID NO:1, oligonucleotides that flankthe extracellular region of LDCAM were used as primers in a PCR reactionto obtain a PCR product from the WI-26 clone. The primers are shown inSEQ ID NO:5 and SEQ ID NO:6. The resulting PCR product was digested withSal1 and Bg1II restriction enzymes at the Sal1 and Bg1II sitesincorporated by the primers. The resulting fragment was ligated into anexpression vector (pDC409) containing the human IgG1 Fc region mutatedto lower Fc receptor binding.

The resulting DNA construct was transfected into the monkey kidney celllines CV-1/EBNA. After 7 days of culture in medium containing 0.5% lowimmunoglobulin bovine serum, a solution of 0.2% azide was added to thesupernatant and the supernatant was filtered through a 0.22 μm filter.Then approximately 1 L of culture supernatant was passed through aBioCad Protein A HPLC protein purification system using a 4.6×100 mmProtein A column (POROS 20A from PerSeptive Biosystems) at 10 mL/min.The Protein A column binds the Fc Portion of the fusion protein in thesupernatant, immobilizing the fusion protein and allowing othercomponents of the supernatant to pass through the column. The column waswashed with 30 mL of PBS solution and bound fusion protein was elutedfrom the HPLC column with citric acid adjusted to pH 3.0. Elutedpurified fusion protein was neutralized as it eluted using IM HEPESsolution at pH 7.4.

EXAMPLE 10 Monoclonal Antibodies to LDCAM

This example illustrates a method for preparing monoclonal antibodies toLDCAM. Purified LDCAM, a fragment thereof such as the extracellulardomain, synthetic peptides or cells that express LDCAM can be used togenerate monoclonal antibodies against LDCAM using conventionaltechniques, for example, those techniques described in U.S. Pat. No.4,411,993. Briefly, mice are immunized with LDCAM as an immunogenemulsified in complete Freund's adjuvant, and injected in amountsranging from 10-100 μg subcutaneously or intraperitoneally. Ten totwelve days later, the immunized animals are boosted with additionalLDCAM emulsified in incomplete Freund's adjuvant. Mice are periodicallyboosted thereafter on a weekly to bi-weekly immunization schedule. Serumsamples are periodically taken by retro-orbital bleeding or tail-tipexcision to test for LDCAM antibodies by dot blot assay or ELISA(Enzyme-Linked Immunosorbent Assay).

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of LDCAM in saline. Three tofour days later, the animals are sacrificed, spleen cells harvested, andspleen cells are fused to a murine myeloma cell line, e.g., NS1 orpreferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified B7L-1 by adaptations of the techniques disclosed in Engvall etal., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004. A preferredscreening technique is the antibody capture technique described inBeckmann et al., (J. Immunol. 144:4212, 1990) Positive hybridoma cellscan be injected intraperitoneally into syngeneic BALB/c mice to produceascites containing high concentrations of anti-LDCAM monoclonalantibodies. Alternatively, hybridoma cells can be grown in vitro inflasks or roller bottles by various techniques. Monoclonal antibodiesproduced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to B7L-1.

EXAMPLE 11 Detecting LDCAM Expression By Northern Blot Analyses

The following describes Northern Blot experiments carried out toidentify tissue and cell types that express LDCAM polypeptides of thepresent invention.

Northern blots were generated by fractionating 5 μg to 10 μg of totalRNA on a 1.2% agarose formaldehyde gel and blotting the RNA onto HybondNylon membranes (Amersham, Arlington Heights, Ill.). Standard northernblot generating procedures as described in Maniatis, (Molecular Cloning:a Laboratory Manual, Cold Spring Harbor Lab. Press, 1989) were used.Poly A+ multiple tissue blots containing 1 μg of mRNA from a number ofdifferent sources were purchased from Clonetech.

A riboprobe, containing the coding region of LDCAM, was generated usingPromega's Riboprobe Combination Kit and T7 RNA Polymerase according tothe manufacturer's instruction. The results of probing the Northernblots and visualizing the resulting x-ray film for positively bindingprobes show that a 5.0 kB hybridizing mRNA was detected for murine LDCAMin lung, liver, brain, testes and splenic dendritic cells. Additionalhybridizing mRNA having different sizes included an approximately 1.9 kBmRNA in lung and testes; an approximately 3.0 kB mRNA in LPS stimulatedbone marrow macrophages, lung and testes; an approximately 7.0 kBhybridizing mRNA in anti-T cell receptor antibody stimulated splenic Tcells, LPS stimulated bone marrow macrophages, and testes; and, anapproximately 9.0 kB hybridizing mRNA was detected in thymus and anti-Tcell receptor antibody stimulated splenic T cells.

EXAMPLE 12 Immune System Cell Binding Studies

The following describes FACS cell binding experiments that demonstratethat LDCAM binds to certain activated immune system cells. For study andcomparison purposes, the binding characteristics of B7L-1 are alsoincluded. Cells studied included murine T cells, human T cells, murine Bcells, murine NK cells, human endothelial cells, and human tumor celllines.

To study murine T cell binding, BALB/c murine lymph node (LN) cells werecultured in culture medium alone and in the presence of differentstimuli for 18-20 hours. The cultured cells were harvested and preparedfor binding studies using B7L1I/Fc fusion protein, LDCAM/Fc fusionprotein and a control Fc protein. Following an overnight culture BALB/cmurine LN cells are typically >90% CD3+. Bound protein was detectedusing flow cytometric analysis. The results shown in Table I indicateobserved binding expressed as mean fluorescence intensity units (MFI) onunstimulated T cells (medium) and on stimulated T cells (by stimuli).

TABLE I Fc medium Con A TCR mAb PHA control Fc 12.7 10.4 14.5 14.2B7L1Fc 11.7 14.3 24.0 12.6 LDCAM Fc 18.7 51.7 230.0 91.4When analyzed by T cell subsets, 75-80% of LN CD4+ murine T cellsdisplayed detectable LDCAM binding after anti-TCR stimulation in vitro.About 50% of LN CD8+ murine T cells display detectable binding. Inaddition, CD4+ T cells exhibit higher levels of LDCAM binding than doCD8+ murine T cells. The results demonstrate that LDCAM/Fc binds at lowlevels to naive T cells. However, after an overnight activation withpolyclonal stimuli binding increased 5-20 fold depending on the stimuli.Of the stimuli studied PMA induces the least LDCAM binding to murine Tcells, and anti-TCR induces the highest binding.

To study human T cells binding to LDCAM and it counterstructure B7L1,human peripheral blood (PB) T cells were cultured in culture medium onlyor in the presence of different stimuli for 18-20 hours. The culturedcells were harvested and prepared for binding studies using eitherB7L/1Fc fusion protein, LDCAM/Fc fusion protein and a control Fcprotein. Bound protein on the human PB T cells was determined by flowcytometric analysis. Table II details results observed, expressed asMFI, on unstimulated T cells (medium) and on stimulated T cells( bystimuli).

TABLE II Fc medium Con A PMA PHA control Fc 4.7 4.8 3.5 4.3 B7L1Fc 6.37.5 4.5 5.7 LDCAM Fc 22.3 42.8 61.9 38.8The results show that, PMA induces greater LDCAM binding on human Tcells than it does on murine T cells. The presence of specific bindingof LDCAM to both murine and human T cells in the absence of B7L1 bindingsuggests that LDCAM is binding to B7L1, or a different molecule and notto itself. Because studies indicate that T cells express little or noB7L1, LDCAM may have another binding partner.Studies similar to those described above were performed to evaluateLDCAM and B7L1 binding to murine splenic B cells. Neither B7L1 nor LDCAMbinding was detected on unstimulated murine B cells. Culturing murinesplenic B cells with muCD40L or LPS induced low levels of LDCAM bindingbut no appreciable level of B7L1 binding was detected.In order to study binding to murine NK cells, spleens were removed fromIL-15 treated CB-17/SCID mice and used as a source for highly enrichedand activated murine NK cells. Spleen cells isolated from IL-15 treatedSCID mice are 60-80% DX-5 positive. DX-5 is a pan NK marker than isexpressed on NK cells from many different strains of mice. Flowcytometric analysis was performed as described above to detect B7L1 andLDCAM binding to DX-5+ in vivo IL-15 activated murine NK cells. Table IIgives the results of a binding murine NK cell binding study.

TABLE III Fc molecule DX-5+ NK cell %+/MFI control Fc 8%/88 B7L1Fc19%/265 LDCAM Fc 38%/432

In contrast to that which was observed on murine and human T cells,LDCAM and B7L1 binding can be detected on in vivo activated murine NKcells.

Results of experiments directed at studying B7L1 and LDCAM binding tohuman endothelial cells demonstrated no binding on human umbilical veinendothelial cells (HUVEC) from different donors. However, one HUVEC fromone donor B7L1 did induce low levels of CD62E and CD106 compared tocontrol Fc.

Table IV details the results of experiments directed at evaluating B7L1and LDCAM binding to human tumor cell lines. The results are expressedas percentage of cells binding LDCAM or B7L1.

TABLE IV LDCAMFc Cell line Cell type (%+)** B7L1Fc (%+)** U937 monocyticleukemia 10 7 K562 erythroblastic 7 5 leukemia Jurkat acute T cellleukemia 10 7 MP-1 B-cell LCL 46 10 DAUDI-hi B-cell Burkitt's 8 6 RPMI8866 B-cell lymphoma 0 0 #88EBV B-cell LCL 4 3 #33EBV B-cell LCL 0 0Tonsil G EBV B-cell LCL 25 13 MDA231 breast 8 9 adenocarcinoma OVCAR-3ovarian carcinoma 48 30 H2126M1 lung adenocarcinoma 0 0 **binding ofcontrol Fc has been subtracted out so this is net %+ cells binding overbackgroundThe results show significant LDCAM binding on ovarian carcinoma cellline and 2 of the human B-cell tumor lines (MP-1 and Tonsil G). B7L1also binds to these three tumor cell lines but a much lower levels.These results demonstrate that LDCAM is a marker for certain types of Bcell lymphomas or different types of carcinomas. In addition, biologicalsignaling mediated by LDCAM or B7L1 could mediate functional anti tumoreffects on these types of tumors.

EXAMPLE 13 Effects of LDCAM on T Cell Proliferation

The following discussion describes experiments performed to evaluate theeffects of LDCAM on murine and human T cell proliferation induced bypolyclonal stimuli.

LDCAM/Fc fusion protein and B7L1/Fc fusion protein were evaluated in astandard model of in vitro murine T cell proliferation. Lymph node (LN)cells were obtained from normal BALB/c mice and placed in culture inmedia. Varying amounts of control Fc, B7L1/Fc and LDCAM/Fc alone or inthe presence of different polyclonal stimuli for T cells including ConA,PHA or immobilized TCR mAb were placed in the culture media.

The results of these experiments demonstrated that LDCAM stronglyinhibits ConA induced murine T cell proliferation (50% inhibition at˜0.625 ug/ml), moderately inhibits PHA induced proliferation ( 50%inhibition at ˜5 ug/ml) and does not effect the proliferation induced byimmobilized TCR mAb. In human peripheral blood T cell proliferationassays, LDCAM inhibits ConA induced proliferation but does noteffectively inhibit PHA or OKT3-induced proliferation. B7L1/Fc does noteffect the proliferative responses of murine or human T cells.

Results suggest that the inhibitory effects of LDCAM/Fc onmitogen-induced murine and human T cell proliferation are due toinhibition of cytokine secretion (especially IL-2) or due to regulationof downstream responses of the T cell following activation and increasesin the expression of the LDCAM binding partner. LDCAM may also modulatecell to cell interactions between T cells, T cells and APC or T cellsand NK cells. The inability of LDCAM to inhibit TCR mAb inducedproliferation suggests that cytokine dysregulation is occurring in thatproliferation induced by ConA and PHA is very cytokine dependent whereas that induced by anti TCR mAb is less so.

EXAMPLE 14 Effects of LDCAM on Murine T Cell Cytokine Production

The following describes experiments performed in order to evaluate LDCAMfor its effects on murine LN cell or purified T cell cytokine secretionfollowing the in vivo activation of T cells with PHA, ConA and TCR mAb.Results are shown in Table V. The levels of cytokine detected areexpressed in pg/ml.

TABLE V culture condition Fc molecule IL-2(pg/ml) IFN-gamma(pg/ml) medianone <2 <10 control Fc <2 <10 LDCAM/Fc <2 <10 ConA none 366 100 controlFc 614 244 LDCAM/Fc <2 <10 PHA none 36 358 control Fc 39 354 LDCAM/Fc 10<10 immob. TCR mAb none 1703 1114 control Fc 1722 1215 LDCAM/Fc 16421027The results show that LDCAM/Fc significantly inhibits murine LN T cellIL-2 and IFN-gamma production that is induced by both ConA and PHA. Whenimmobilized anti TCR mAb is used to induce cytokine production frommurine T cell, less pronounced effects of LDCAM on cytokine productionwere observed. LDCAM decreased IFN-gamma production after TCRactivation. In contrast, IL-2 production was not decreased after TCRactivation. Very little IL-4 was generated by the T cells in theseexperiments so whether or not LDCAM effects T cell production of IL-4 orother additional cytokines/chemokines was not evaluated.

EXAMPLE 15 Effects of LDCAM on Murine Mixed Cell Activation Assays

An in vitro mixed cell assay was developed to examine the ability of Tcells to activate B cells through their CD40L/CD40 interaction. Theassay involves culturing spleen cells and LN cells with anti-TCR mAb invitro for 36 hours followed by the flow cytometric analysis T and Bcell/APC cell activation that occurs after T cells become activated andinteract with B cells/APCs.

Spleen cells were cultured with anti TCR mAb, ConA, PHA or in media onlywith control Fc or LDCAM/Fc for 36 hours. CD19+ B cell and CD3+ T cellactivation was followed by examining cell surface expression of CD25,CD69, CD54, CD45Rb, CD44, CD28, CD23, CD86 and CD152 using two-colorstaining and flow cytometric analysis.

The results demonstrated that after activation with PHA or ConA theexpression of CD69, CD54, and CD25 increases several fold on T cells andB cells in the culture. Compared to a control Fc which has little effecton these increases, LDCAM significantly reduced expression (almost tothe same levels as non-activated T cells) of CD69, CD54 and CD25 thatare induced on both cells types in this culture system via activationwith ConA. The ConA activates the T cells which express activationmolecules (e.g. CD40L) on their surface. The activation molecules bindto receptors on the surface of B cells and activate the B cells toexpress various activation-related proteins on their cell surface. Theinhibition PHA activated T and B cells occurred to a more moderateextent to that observed after activation with ConA.

In addition, LDCAM decreased the levels of CD45RB expressed on both CD3+and CD3− in spleen cells cultured with ConA. This effect on decreasingCD45RB levels was more pronounced when LDCAM was cultured with spleencells stimulated with TCR mAb and was not observed when PHA was used asa stimulus or when the cells were cultured in medium alone.

Using TCR mAb to stimulate the cultured spleen cells in the presence ofa control Fc or LDCAM/Fc showed that the levels of CD69, CD25, and CD25induced on T cells and B cells by this stimulus were not effected byLDCAM. However, LDCAM increased the expression of CD28 on both CD3+ Tcells and non-T cells. In one experiment the increase was 5-10 fold andin the other experiments the increase was 50%. This was also observed inone experiment when ConA was used as a stimulus in addition to TCR mAb.LDCAM caused moderate decreases in the intensity of CD45RB expression onB cells (50% decrease) and T cells(20-30% decrease) after activationwith TCR mAb.

Interestingly, LDCAM does not effect CD45RB expression on spleen cellswhen they are cultured in the absence of polyclonal T cell stimuli.CD45RB expression in rodents has been reported to decrease as T cellsprogress from naive to memory cells. Also different subpopulations ofCD4+ T cells express high or low levels of CD45RB and mediate distinctimmune functions in vivo.

The above discussed results suggest that under certain immunestimulation conditions, particularly stimulations by ConA and PHA, LDCAMinhibits T cell activation at the cellular level in mixed cell assaysand inhibits T cell proliferation induced by these mitogens at leastpartially by decreasing IL-2 and IFN-gamma production.

While LDCAM modestly down-regulates IFN gamma production induced by TCRmAb-induced activation, it has little effect on IL-2 production in thissystem and does not effect proliferation of murine T cells induced byimmobilized TCR mAb. LDCAM does cause an increase in the TCR mAbactivated T-cell and B-cell expression of CD28 and a decrease in CD45RBexpression. Based on these data, LDCAM or its binding partner on T cellscan regulate (increase, decrease or redirect) T cell effector-dependentimmune responses in vivo including but not limited to anti-tumor immuneresponses, DTH responses, and T-cell dependent anti-infectious diseaseimmune responses.

The above results suggest that LDCAM is useful in modulating T cellactivation pathways and can be used to treat autoimmune diseases andinflammation.

EXAMPLE 16 LDCAM.Fc Binds to Murine NK Cells and Causes NK CellExpansion

The following describes experiments that demonstrate that LDCAM binds tothe surface of splenic NK cells constituitively and that activation ofthese cells with IL-15 increased the levels of LDCAM binding. Theexperiments also describes administering LDCAM:Fc to CB-17 SCID mice andthe effects of the administration on NK cell expansion and activation inthe spleen.

Twelve age-matched female CB-17/SCID mice were divided into 4 groups,with 3 animals per group. On day O, day 1 and day 2, group I, group II,group III and group IV were administered the following proteins IP:group I mice received 10 μg of human IgG; group II mice received 10 μgof human IL-15; group III mice received 10 μg of human LDCAM:Fc (lot#7488-16 from Immunex); and, group IV received 10 μg each of humanLDCAM:Fc and human IL-15.

On day 3 (the 4^(th) day of the experiment), the mice were euthanizedand their spleens were removed. Each spleen was enumerated separatelyand then pooled together for flow cytometric analysis. The number of NKcells in the spleen of each treated group was determined by flowcytometry using the DX-5 antibody as a pan-murine NK cell marker. Inaddition, other measures of NK cell activation including CD69 and CD54expression were evaluated.

The results for the experiment are shown in Table VI. Administration ofLDCAM:Fc alone (Group III) increased the total recovered spleen cellnumber by about 5-fold over the human IgG control group (Group I).Administration of human IL-15 alone, (Group II) increased the totalrecovered spleen cell number by about 9-fold over the control group(Group I). Combination treatment with IL-15 and LDCAM increased thespleen cell number additively.

The number of NK cells recovered from the spleens correlated with thetotal cell recovery in the spleen. More particularly, LDCAM inducedabout a 5-fold increase in recovered NK cells; IL-15 caused about a9-fold increase in recovered NK cells; and, the combination of LDCAM andIL-15 induced about a 13-fold increase in the number of NK cellsrecovered from the spleens of treated mice. LDCAM also increased thenumber of NK cells in the spleen that expressed CD69 and CD54. Thisincrease was due to overall NK cell expansion rather than specificincreases in the expression of CD69 or CD54 on NK cells in vivofollowing LDCAM:Fc administration.

TABLE VI % DX-5⁺ # of NK cells spleen cell Number cells recovered × SCIDMice Group counts × 10⁶ of Mice (NK) 10⁶ Group I (human IgG 2.3 3 67.81.6 control) Group II (IL15 17.8 3 81.7 14.5 positive control) Group III10.25 3 51.2 5.3 (LDCAM:Fc) LDCAM:Fc and 24.8 3 72.6 18.0 IL15

1. An isolated antibody that specifically binds to SEQ ID NO:2.
 2. Anisolated antibody that specifically binds to SEQ ID NO:4.
 3. Theantibody of claim 1, wherein the antibody is a monoclonal antibody. 4.The antibody of claim 1, wherein the antibody specifically binds to theextracellular domain of SEQ ID NO:2, wherein the extracellular domaincomprises amino acids 39 to 374 of SEQ ID NO:2.
 5. The antibody of claim4, wherein the antibody is a monoclonal antibody.
 6. A hybridoma thatproduces the antibody of claim 5.